Heparan Sulfate Proteoglycan Syndecan-1 Research Articles

Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro. The heparan sulfate proteoglycan syndecan-4 regulates cell migration and is crucial for wound healing. We report that syndecan-4 expression is restricted to epidermis and blood vessels in mice skin wounds. Syndecan-4 regulates binding and internalisation of CAR peptide and CAR-mediated cytoskeletal remodelling. CAR induces syndecan-4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor cytohesin-2, and promotes syndecan-4-, ARF6- and Cytohesin-2-mediated keratinocyte migration. Finally, we show that genetic ablation of syndecan-4 in male mice eliminates CAR-induced wound re-epithelialisation following systemic administration. We propose that CAR peptide activates syndecan-4 functions to selectively promote re-epithelialisation. Thus, CAR peptide provides a therapeutic approach to enhance wound healing in mice; systemic, yet target organ- and cell-specific.

THE IMPACT OF SEX ON BONE STRUCTURE AND ANGIOGENESIS IN SYNDECAN-1 DEFICIENT MICE

Neoangiogenesis drives the replacement of mineralised cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL. The Heparan sulfate proteoglycan Syndecan-1 (Sdc1) plays a role in the interaction of osteoclasts and osteoblasts and the development of blood vessels. We expected Sdc1 to have an influence on bone structure and vessel development. Therefore, bone structure and angiogenesis at the growth plate in mice was compared and the influence of Syndecan-1 deficiency was characterised.Animals: Femura of male and female C57BL/6 WT (5♀, 6♂) and Sdc1-/- (9♀, 5♂) mice were used for native bone analysis at 4 month age. Histology: Bone structure was analysed using microCT scans with a resolution of 9µm. Vascularisation was visualised using an anti-Endomucin antibody in 80µm thick cryosections. In vitro angiogenesis: Bone marrow isolates were used to generate endothelial progenitor cells by sequential cultivation on fibronectin. Microvessel development was analysed 4h after plating on matrigel.Bone structure in male Sdc1 deficient mice was significantly reduced compare to male WT, whereas female mice of both genotypes did not differ. Sdc1 deficient mice at the age of 4 month showed a high decrease in the number of vessel bulbs at the chondro-osseous border (growth plate) compared to WT mice. However, no sex related differences were shown. Quantification of microvessel outgrowth of endothelial cells revealed a decreased amount of sprouting, but increased length of microvessels of Sdc1-/- cells compared to WT.Syndecan-1 has a significant impact on neoangiogenesis at the chondro-osseous border of the native bone, but the impact of Syndecan-1 deficiency on the loss of bone structure was significantly higher in male mice. This emphasises the importance to further characterise the function of Syndecan-1 regulated processes during enchondral ossification in a sex dependent manner.

The Heparan Sulfate Proteoglycan Syndecan-1 Triggers Breast Cancer Cell-Induced Coagulability by Induced Expression of Tissue Factor.

Syndecan-1 (Sdc-1) upregulation is associated with poor prognosis in breast cancer. Sdc-1 knockdown results in reduced angiogenesis and the dysregulation of tissue factor (TF) pathway constituents. Here, we evaluate the regulatory mechanisms and functional consequences of the Sdc-1/TF-axis using Sdc-1 knockdown and overexpression approaches in MCF-7 and MDA-MB-231 breast cancer cells. Gene expression was analyzed by means of qPCR. Thrombin generation and cell migration were detected. Cell-cycle progression and apoptosis were investigated using flow cytometry. In MDA-MB-231 cells, IL6, IL8, VEGF, and IGFR-dependent signaling affected TF pathway expression depending on Sdc-1. Notably, Sdc-1 depletion and TF pathway inhibitor (TFPI) synergistically affected PTEN, MAPK, and STAT3 signaling. At the functional level, the antiproliferative and pro-apoptotic effects of TFPI depended on Sdc-1, whereas Sdc-1's modulation of cell motility was not affected by TFPI. Sdc-1 overexpression in MCF-7 and MDA-MB-231 cells led to increased TF expression, inducing a procoagulative phenotype, as indicated by the activation of human platelets and increased thrombin formation. A novel understanding of the functional interplay between Sdc-1 and the TF pathway may be compatible with the classical co-receptor role of Sdc-1 in cytokine signaling. This opens up the possibility of a new functional understanding, with Sdc-1 fostering coagulation and platelet communication as the key to the hematogenous metastatic spread of breast cancer cells.

Role of syndecan-4 in breast cancer pathophysiology.

Expression of the cell surface heparan sulfate proteoglycan syndecan-4 is dysregulated in breast cancer, the most frequent malignancy in women. High expression of syndecan-4 correlates with a worse survival in the subgroup of estrogen receptor negative and estrogen/progesterone-receptor negative patients. Aberrant expression of syndecan-4 in breast cancer involves both transcriptional and posttranscriptional mechanisms, including estrogen- and growth factor-dependent regulation, mutations in GAPVD1, NUP153, PDE4DIP, and RREB1, as well as targeting by microRNAs. At the functional level, syndecan-4 plays an important role in various stages of breast cancer progression by interacting with ligands as diverse as plasma proteins, extracellular matrix proteins, growth factors, and surface receptors, as well as members of the integrin family. Mechanisms including integrin recycling, ectodomain shedding, and crosstalk with other syndecans expand the repertoire of syndecan-4 function. Through these interactions, syndecan-4 regulates cellular processes such as adhesion, migration, and invasion. Additional possible functions of syndecan-4 in cells of the microenvironment contribute to the complexity of its pathophysiology. Notably, syndecan-4 expression is modulated by drugs used in breast cancer treatment, such as trastuzumab and zoledronate. Overall, these findings mark syndecan-4 as a novel pathogenesis factor and promising target for therapeutic interventions in breast cancer.

Roles of Syndecan-4 in cardiac injury and repair.

The heparan sulphate proteoglycan Syndecan-4 belongs to a 4-member family of transmembrane receptors. Genetic deletion of Syndecan-4 in mice causes negligible developmental abnormalities however when challenged these animals show distinct phenotypes. Synedcan-4 is expressed in many cell types in the heart and its expression is elevated in response to cardiac injury and recent studies have suggested roles for Syndecan-4 in repair mechanisms within the damaged heart. The purpose of this review is to explore these biological insights into the role of Syndecan-4 in both the injured heart and later during cardiac repair and remodeling.

Differential Impact of Membrane-Bound and Soluble Forms of the Prognostic Marker Syndecan-1 on the Invasiveness, Migration, Apoptosis, and Proliferation of Cervical Cancer Cells.

Cervical cancer ranks fourth among the most commonly diagnosed malignant tumors in women worldwide. Previously published evidence suggested a possible connection between the expression of the membrane-bound heparan sulfate proteoglycan syndecan-1 (Sdc-1) and the development of cervical carcinoma. Sdc-1 serves as a matrix receptor and coreceptor for receptor tyrosine kinases and additional signaling pathways. It influences cell proliferation, adhesion, and migration and is seen as a modulator of the tumor microenvironment. Following proteolytic cleavage of its extracellular domain in a process called shedding, Sdc-1 can act as a paracrine effector. The loss of Sdc-1 expression is associated with low differentiation of cervical carcinoma and with an increased rate of lymph node metastases. Here, we analyzed the clinical impact of Sdc-1 expression by analysis of public gene expression datasets and studied the effect of an overexpression of Sdc-1 and its membrane-bound and soluble forms on the malignant properties of the human cervical carcinoma cell line HeLa through functional analysis. For this purpose, the HeLa cells were stably transfected with the control plasmid pcDNA3.1 and three different Sdc-1-DNA constructs,encoding wild-type, permanently membrane-bound, and constitutively soluble Sdc-1. In clinical specimens, Sdc-1 mRNA was more highly expressed in local tumor tissues than in normal and metastatic cervical cancer tissues. Moreover, high Sdc-1 expression correlated with a poor prognosis in Kaplan-Meier survival analysis, suggesting the important role of Sdc-1 in the progression of this type of cancer. In vitro, we found that the soluble, as well as the permanently membrane-bound forms of Sdc-1 modulated the proliferation and the cell cycle, while membrane-bound Sdc1 regulated HeLa cell apoptosis. The overexpression of Sdc-1 and its soluble form increased invasiveness. In vitro scratch/wound healing assay, showed reduced Sdc-1-dependent cell motility which was linked to the Rho-GTPase signaling pathway. In conclusion, in cervical cancer Sdc-1 modulates pathogenetically relevant processes, which depend on the membrane-association of Sdc-1.

HIV-1 Tat and Heparan Sulfate Proteoglycans Orchestrate the Setup of in Cis and in Trans Cell-Surface Interactions Functional to Lymphocyte Trans-Endothelial Migration.

HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This “two-way” activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.

Heparan sulfate proteoglycans in beta cells provide a critical link between endoplasmic reticulum stress, oxidative stress and type 2 diabetes.

Heparan sulfate proteoglycans (HSPGs) consist of a core protein with side chains of the glycosaminoglycan heparan sulfate (HS). We have previously identified (i) the HSPGs syndecan-1 (SDC1), and collagen type XVIII (COL18) inside mouse and human islet beta cells, and (ii) a critical role for HS in beta cell survival and protection from reactive oxygen species (ROS). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress contributes to oxidative stress and type 2 diabetes (T2D) by depleting beta cell HSPGs/HS. A rapid loss of intra-islet/beta cell HSPGs, HS and heparanase (HPSE, an HS-degrading enzyme) accompanied upregulation of islet ER stress gene expression in both young T2D-prone db/db and Akita Ins2WT/C96Y mice. In MIN6 beta cells, HSPGs, HS and HPSE were reduced following treatment with pharmacological inducers of ER stress (thapsigargin or tunicamycin). Treatment of young db/db mice with Tauroursodeoxycholic acid (TUDCA), a chemical protein folding chaperone that relieves ER stress, improved glycemic control and increased intra-islet HSPG/HS. In vitro, HS replacement with heparin (a highly sulfated HS analogue) significantly increased the survival of wild-type and db/db beta cells and restored their resistance to hydrogen peroxide-induced death. We conclude that ER stress inhibits the synthesis/maturation of HSPG core proteins which are essential for HS assembly, thereby exacerbating oxidative stress and promoting beta cell failure. Diminished intracellular HSPGs/HS represent a previously unrecognized critical link bridging ER stress, oxidative stress and beta cell failure in T2D.

Syndecan-1 Promotes Angiogenesis in Triple-Negative Breast Cancer through the Prognostically Relevant Tissue Factor Pathway and Additional Angiogenic Routes.

Simple SummaryTriple-negative breast cancer is an aggressive subtype of breast cancer characterized by tumor angiogenesis and poor patient survival. Here, we analyzed the function of the cell surface molecule Syndecan-1 in tumor angiogenesis in a 3D cell culture system. As a novel finding, we demonstrate that downregulation of Syndecan-1 reduces angiogenesis by decreasing the amount of angiogenesis factors of the tissue factor pathway. Furthermore, we show that the components of this pathway are associated with the prognosis of breast cancer patients. Our study identifies Syndecan-1 and the tissue factor pathway as novel potential therapeutic targets in the aggressive triple-negative subtype of breast cancer, for which no targeted therapies are currently available.Triple-negative breast cancer (TNBC) is characterized by increased angiogenesis, metastasis, and poor survival. Dysregulation of the cell surface heparan sulfate proteoglycan and signaling co-receptor Syndecan-1 is linked to poor prognosis. To study its role in angiogenesis, we silenced Syndecan-1 in TNBC cell lines using a 3D human umbilical vein endothelial cell (HUVEC) co-culture system. Syndecan-1 siRNA depletion in SUM-149, MDA-MB-468, and MDA-MB-231 cells decreased HUVEC tubule network formation. Angiogenesis array revealed reduced VEGF-A and tissue factor (TF) in the Syndecan-1-silenced secretome. qPCR independently confirmed altered expression of F3, F7, F2R/PAR1, F2RL1/PAR2, VEGF-A, EDN1, IGFBP1, and IGFBP2 in SUM-149, MDA-MB-231, and MDA-MB-468 cells. ELISA revealed reduced secreted endothelin-1 (SUM-149, MDA-MB-468) and TF (all cell lines) upon Syndecan-1 depletion, while TF pathway inhibitor treatment impaired angiogenesis. Survival analysis of 3951 patients demonstrated that high expression of F3 and F7 are associated with better relapse-free survival, whereas poor survival was observed in TNBC and p53 mutant basal breast cancer (F3) and in ER-negative and HER2-positive breast cancer (F2R, F2RL1). STRING protein network analysis revealed associations of Syndecan-1 with VEGF-A and IGFBP1, further associated with the TF and ET-1 pathways. Our study suggests that TNBC Syndecan-1 regulates angiogenesis via the TF and additional angiogenic pathways and marks its constituents as novel prognostic markers and therapeutic targets.

Circulating Levels of the Heparan Sulfate Proteoglycan Syndecan-4 Positively Associate with Blood Pressure in Healthy Premenopausal Women.

Syndecans (SDCs) are transmembrane proteins that are present on most cell types where they play a role in multiple physiological processes, including cell–matrix adhesion and inflammation. Growing evidence suggests that elevated levels of both shed SDC1 and SDC4 are associated with hypertension and cardiovascular diseases, but their relationships with cardiovascular risk factors in healthy individuals are unknown. The primary objective of this study was to investigate whether serum levels of SDC4 and SDC1 were associated with body composition, hemodynamic parameters, pro-inflammatory cytokine concentrations, and urinary noradrenaline and dopamine levels in healthy women (17 African American and 20 European American) between the ages of 20 and 40 years old. Univariate analyses revealed only a significant (p < 0.05) inverse correlation between serum SDC1 and body fat percentage. On the other hand, serum SDC4 was positively correlated with systolic blood pressure, diastolic blood pressure, and urinary levels of noradrenaline and dopamine. Serum SDC4 was also a significant predictor of systolic blood pressure in a multivariate regression model that included fat-free mass and urinary dopamine levels as significant independent variables. The result did not change even adjusting for race. Our findings indicate that SDC4 has an important role in the physiological regulation of blood pressure.

Convergent chemoenzymatic synthesis and biological evaluation of a heparan sulfate proteoglycan syndecan-1 mimetic.

A new convergent chemoenzymatic synthesis strategy, integrating enzymatic synthesis of heparan sulfate, sortase A ligation, copper(i)-catalyzed alkyne-azide cycloaddition, and solid phase peptide synthesis, has been established to efficiently synthesize a mimetic of heparan sulfate proteoglycan syndecan-1 glyco-polypeptide at a milligram scale. The mimic was able to bind with αvβ3 integrin faster and exhibit stronger inhibition of breast cancer cell migration compared to the glycan or the polypeptide alone. This novel approach could serve as a general approach for heparan sulfate proteoglycan mimetic synthesis.

Expression of Syndecan-1 in Chronic Liver Diseases: Correlation With Hepatic Fibrosis.

The mechanisms underlying the contribution of the heparan sulfate proteoglycan syndecan-1 to liver tissue injury and to crucial biological processes, such as fibrogenesis, remain to be elucidated. Therefore, we investigated the immunohistochemical expression of syndecan-1 in chronic liver diseases (CLDs) and its probable role in hepatic fibrosis. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections of biopsy material obtained from 128 patients diagnosed with CLDs. The correlation between syndecan-1 expression and the stage of fibrosis was investigated. According to the severity of fibrosis, cases were categorized into three groups: early fibrosis; intermediate fibrosis; advanced fibrosis. Syndecan-1 expression was significantly enhanced in advanced fibrosis compared to early (p<0.012) and intermediate (p<0.003) fibrosis. In CLDs, syndecan-1 immunohisto-chemical overexpression was found to be positively correlated with the severity of fibrosis, suggesting its probable role in hepatic fibrogenesis.

The α-defensins stimulate proteoglycan-dependent catabolism of low-density lipoprotein by vascular cells: a new class of inflammatory apolipoprotein and a possible contributor to atherogenesis

AbstractInflammation may contribute to the pathogenesis of atherosclerosis. On the basis of previous reports that human atherosclerotic lesions contain α-defensins, a class of cationic proteins released by activated neutrophils, the study was designed to ask whether defensins modulate the binding and catabolism of low-density lipoprotein (LDL) by human vascular cells. The results of the study demonstrated that defensin stimulated the binding of 125I-LDL to cultured human umbilical vein endothelial cells, smooth muscle cells, and fibroblasts approximately 5-fold in a dose-dependent and saturable manner. Defensin and LDL formed stable complexes in solution and on cell surfaces. Stimulation of LDL binding by defensin was not inhibited by antibodies against the LDL-receptor (LDL-R), or by recombinant receptor-associated protein, which blocks binding of ligands to the α2-macroglobulin receptor/LDL-R–related protein and other LDL-R family members. Furthermore, defensin stimulated the binding, endocytosis, and degradation of LDL by fibroblasts lacking LDL-R. Stimulation of LDL degradation by defensin was inhibited approximately 75% by low concentrations of heparin (0.2 units/mL) and was similarly reduced in CHO cells lacking heparan-sulfate–containing proteoglycans. The effect of defensin was substantially increased in cells overexpressing the core protein of the syndecan-1 heparan sulfate proteoglycan. The α-defensins released from activated neutrophils may provide a link between inflammation and atherosclerosis by changing the pattern of LDL catabolism from LDL-R to the less efficient LDL-R–independent, proteoglycan-dependent pathway.

The heparan sulfate proteoglycan Syndecan-1 influences local bone cell communication via the RANKL/OPG axis

The heparan sulfate proteoglycan Syndecan-1, a mediator of signals between the extracellular matrix and cells involved is able to interact with OPG, one of the major regulators of osteoclastogenesis. The potential of osteoblasts to induce osteoclastogenesis is characterized by a switch of OPG (low osteoclastogenic potential) towards RANKL production (high osteoclastogenic potential). In the present study, we investigated the influence of endogenous Syndecan-1 on local bone-cell-communication via the RANKL/OPG-axis in murine osteoblasts and osteoclasts in wild type and Syndecan-1 lacking cells. Syndecan-1 expression and secretion was increased in osteoblasts with high osteoclastogenic potential. Syndecan-1 deficiency led to increased OPG release by osteoblasts that decreased the availability of RANKL. In co-cultures of Syndecan-1 deficient osteoblasts with osteoclast these increased OPG in supernatant caused decreased development of osteoclasts. Syndecan-1 and RANKL level were increased in serum of aged WT mice, whereas Syndecan-1 deficient mice showed high serum OPG concentration. However, bone structure of Syndecan-1 deficient mice was not different compared to wild type. In conclusion, Syndecan-1 could be regarded as a new modulator of bone-cell-communication via RANKL/OPG axis. This might be of high impact during bone regeneration or bone diseases like cancer where Syndecan-1 expression is known to be even more prevalent.

Shiga Toxin Selectively Upregulates Expression of Syndecan-4 and Adhesion Molecule ICAM-1 in Human Glomerular Microvascular Endothelium.

Hemolytic uremic syndrome (HUS) is a severe renal disease that is often preceded by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). The exact mechanism of Stx-mediated inflammation on human glomerular microvascular endothelial cells (HGMVECs) during HUS is still not well understood. In this study, we investigated the effect of Stx1 on the gene expression of proteins involved in leucocyte-mediated and complement-mediated inflammation. Our results showed that Stx1 enhances the mRNA and protein expression of heparan sulfate proteoglycan (HSPG) syndecan-4 in HGMVECs pre-stimulated with tumor necrosis factor α (TNFα). CD44 was upregulated on mRNA but not on protein level; no effect on the mRNA expression of other tested HSPGs glypican-1 and betaglycan was observed. Furthermore, Stx1 upregulated the mRNA, cell surface expression, and supernatant levels of the intercellular adhesion molecule-1 (ICAM-1) in HGMVECs. Interestingly, no effect on the protein levels of alternative pathway (AP) components was observed, although C3 mRNA was upregulated. All observed effects were much stronger in HGMVECs than in human umbilical endothelial cells (HUVECs), a common model cell type used in endothelial studies. Our results provide new insights into the role of Stx1 in the pathogenesis of HUS. Possibilities to target the overexpression of syndecan-4 and ICAM-1 for STEC-HUS therapy should be investigated in future studies.

Inhibition of Heparanase Expression Results in Suppression of Invasion, Migration and Adhesion Abilities of Bladder Cancer Cells.

Heparan sulfate proteoglycan syndecan-1, CD138, is known to be associated with cell proliferation, adhesion, and migration in malignancies. We previously reported that syndecan-1 (CD138) may contribute to urothelial carcinoma cell survival and progression. We investigated the role of heparanase, an enzyme activated by syndecan-1 in human urothelial carcinoma. Using human urothelial cancer cell lines, MGH-U3 and T24, heparanase expression was reduced with siRNA and RK-682, a heparanase inhibitor, to examine changes in cell proliferation activity, induction of apoptosis, invasion ability of cells, and its relationship to autophagy. A bladder cancer development mouse model was treated with RK-682 and the bladder tissues were examined using immunohistochemical analysis for Ki-67, E-cadherin, LC3, and CD31 expressions. Heparanase inhibition suppressed cellular growth by approximately 40% and induced apoptosis. The heparanase inhibitor decreased cell activity in a concentration-dependent manner and suppressed invasion ability by 40%. Inhibition of heparanase was found to suppress autophagy. In N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer mice, treatment with heparanase inhibitor suppressed the progression of cancer by 40%, compared to controls. Immunohistochemistry analysis showed that heparanase inhibitor suppressed cell growth, and autophagy. In conclusion, heparanase suppresses apoptosis and promotes invasion and autophagy in urothelial cancer.

The heparan sulfate proteoglycan syndecan‐1 regulates colon cancer stem cell function via a focal adhesion kinase—Wnt signaling axis

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of β1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.

Syndecan-1-Dependent Regulation of Heparanase Affects Invasiveness, Stem Cell Properties, and Therapeutic Resistance of Caco2 Colon Cancer Cells.

The heparan sulfate proteoglycan Syndecan-1 binds cytokines, morphogens and extracellular matrix components, regulating cancer stem cell properties and invasiveness. Syndecan-1 is modulated by the heparan sulfate-degrading enzyme heparanase, but the underlying regulatory mechanisms are only poorly understood. In colon cancer pathogenesis, complex changes occur in the expression pattern of Syndecan-1 and heparanase during progression from well-differentiated to undifferentiated tumors. Loss of Syndecan-1 and increased expression of heparanase are associated with a change in phenotypic plasticity and an increase in invasiveness, metastasis and dedifferentiation. Here we investigated the regulatory and functional interplay of Syndecan-1 and heparanase employing siRNA-mediated silencing and plasmid-based overexpression approaches in the human colon cancer cell line Caco2. Heparanase expression and activity were upregulated in Syndecan-1 depleted cells. This increase was linked to an upregulation of the transcription factor Egr1, which regulates heparanase at the promoter level. Inhibitor experiments demonstrated an impact of focal adhesion kinase, Wnt and ROCK-dependent signaling on this process. siRNA-depletion of Syndecan-1, and upregulation of heparanase increased the colon cancer stem cell phenotype based on sphere formation assays and phenotypic marker analysis (Side-population, NANOG, KLF4, NOTCH, Wnt, and TCF4 expression). Syndecan-1 depletion increased invasiveness of Caco2 cells in vitro in a heparanase-dependent manner. Finally, upregulated expression of heparanase resulted in increased resistance to radiotherapy, whereas high expression of enzymatically inactive heparanase promoted chemoresistance to paclitaxel and cisplatin. Our findings provide a new avenue to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence.

ADAMTS-1 and syndecan-4 intersect in the regulation of cell migration and angiogenesis.

ABSTRACTADAMTS-1 is an extracellular protease with critical roles in organogenesis and angiogenesis. Here we demonstrate a functional convergence of ADAMTS-1 and the transmembrane heparan sulfate proteoglycan syndecan-4 in influencing adhesion, migration and angiogenesis. Knockdown of ADAMTS-1 in endothelial cells resulted in a parallel reduction in cell surface syndecan-4, attributable to increased matrix metalloproteinase-9 (MMP9) activity. Knockdown of either ADAMTS-1 or syndecan-4 increased cellular responses to vascular endothelial growth factor A isoform VEGFA164, and increased ex vivo aortic ring microvessel sprouting. On fibronectin, knockdown of either protein enhanced migration and promoted formation of long α5 integrin-containing fibrillar adhesions. However, integrin α5 null cells still showed increased migration in response to ADAMTS-1 and syndecan-4 siRNA treatment. Plating of naïve endothelial cells on cell-conditioned matrix from ADAMTS-1 and syndecan-4 knockdown cells demonstrated that the altered adhesive behaviour was matrix dependent, and this correlated with a lack of expression of fibulin-1: an extracellular matrix co-factor for ADAMTS-1 that is known to inhibit migration. These findings support the notion that ADAMTS-1 and syndecan-4 are functionally interconnected in regulating cell migration and angiogenesis, via collaboration with MMP9 and fibulin-1.This article has an associated First Person interview with the first author of the paper.

Insights into the heparan sulphate-dependent externalisation of transglutaminase-2 (TG2) in glucose-stimulated proximal-like tubular epithelial cells

The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, suggesting that TG2-externalisation depends on an intact TG2 heparin binding site. Inhibition of N-ethylmaleimide sensitive factor (NSF) vesicle-fusing ATPase, which was identified in the recently elucidated TG2 kidney membrane-interactome, led to significantly lower TG2-externalisation, thus validating the involvement of membrane fusion in TG2 secretion. As cyclin-G-associated kinase (GAK) had emerged as a further TG2-partner in the fibrotic kidney, we investigated whether glucose-induced TG2-externalisation was accompanied by TG2 phosphorylation in consensus sequences of cyclin-dependent kinase (CDK). Glucose stress led to intense TG2 phosphorylation in serine/threonine CDK-target. TG2 phosphorylation by tyrosine kinases was also increased by glucose. Although the precise role of glucose-induced TG2 phosphorylation is unknown, these novel data suggest that phosphorylation may be involved in TG2 membrane-trafficking.